Endogenous nitrosation of amino acids and related molecules is an important potential source of carcinogen N-nitroso compounds in human populations. Of the several nitroso compounds (N-nitrososarcosine, N-hydroxynitrosoproline and N-nitrosopyrrolidine, etc.) shown to occur in human urine and food stuffs, NPro is one that is stable, nonmutagenic and noncarcinogenic and can be safely given to humans. Ingested preformed NPro is excreted unchanged in human and animal urine and its concentration in urine increases proportionally to increases in percursor nitrates and L-prolines consumed, respectively, in food stuffs and drinking water. In addition, higher levels of urinary NPro have been found in smokers than in nonsmokers and in individuals residing in geographical areas with high incidence of stomach and esophageal cancer than in persons from areas with low incidence of these diseases. Studies of urinary NPro are currently accomplished by a measure of in vitro methylated urinary NPro (NPro (CH3)) in gas chromatography with a thermal energy analyzer (GC-TEA). We have developed an immunoassay for NPro (CH3) which may simplify and facilitate the study of this potentially important phenomenon. Our rabbit and anti-KLH-NPro antisera does not recognize proline nor unconjugated NPro, but does recognize the methyl ester of NPro (NPro (CH3)) and nitroprolylglycine (NPG). Nine esterfied extracts of human urine previously tested positive in GC-TEA have been tested positive in ELISA, and all measured apparently higher in GC-TEA. Characterization of the antibody is incomplete and the quantitative difference may be due to detection of substances other than NPro. However, NPG is not known to be present in biological materials.